A novel prognostic signature in osteosarcoma characterised from the perspective of unfolded protein response

Dear Editor, Osteosarcoma (OS) is the most common primary malignant tumour of bone with variable molecular biology and prognosis. This makes better patient stratification and precision treatment an urgent clinical need.1 Activation of the unfolded protein response (UPR) is a hallmark of cancer cells facing endoplasmic reticulum (ER) stress,2,3 yet its clinical relevance inOS remains to be explored. By comprehensive interrogation of OS datasets established by us and others,4–7 the present study consolidates UPR activation as a critical molecular feature of OS and refines a prognostic gene signature from this perspective with translational potential. In this study (see Figure S1A for workflow), we assembled 5 independent OS cohorts (GSE99671, GSE126209, GSE21257, TARGET and Zhengzhou datasets), plus the TCGA sarcoma dataset. Three datasets (GSE99671, GSE126209 and Zhengzhou) with paired tumour and normal tissues were analysed for deregulated genes; two of them with relatively large sample size were further selected for pathway enrichment. Three datasets (GSE21257, TARGET and TCGA) with solely tumours and survival information for patient classification and prognostic model construction (Table S1). We first interrogated GSE99671 with paired tumour and normal samples, and identified 1581 differentially expressed genes (DEGs) [|log2 (fold change)| > .5 and adjust p value < .05] (Figure S1B). To our interest, several pathways related to ER function, such as response to ER stress and protein processing in ER, ranked top according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses (Figure 1A and B). Enrichment of UPR and MYC targets was verified by Hallmark Gene Set Enrichement Analysis (GSEA) (Figures 1C and S1C), mirroring the recently established co-activation of UPR and MYC in multiple cancers.4,8 Meanwhile, transcriptomic analyses on 24 matched tumour and normal tissues collected in our hospital (hereafter referred to as Zhengzhou cohort) enriched similar

A novel prognostic signature in osteosarcoma characterised from the perspective of unfolded protein response Dear Editor, Osteosarcoma (OS) is the most common primary malignant tumour of bone with variable molecular biology and prognosis. This makes better patient stratification and precision treatment an urgent clinical need. 1 Activation of the unfolded protein response (UPR) is a hallmark of cancer cells facing endoplasmic reticulum (ER) stress, 2,3 yet its clinical relevance in OS remains to be explored. By comprehensive interrogation of OS datasets established by us and others, 4-7 the present study consolidates UPR activation as a critical molecular feature of OS and refines a prognostic gene signature from this perspective with translational potential.
In this study (see Figure S1A for workflow), we assembled 5 independent OS cohorts (GSE99671, GSE126209, GSE21257, TARGET and Zhengzhou datasets), plus the TCGA sarcoma dataset. Three datasets (GSE99671, GSE126209 and Zhengzhou) with paired tumour and normal tissues were analysed for deregulated genes; two of them with relatively large sample size were further selected for pathway enrichment. Three datasets (GSE21257, TARGET and TCGA) with solely tumours and survival information for patient classification and prognostic model construction (Table S1).
We first interrogated GSE99671 with paired tumour and normal samples, and identified 1581 differentially expressed genes (DEGs) [|log2 (fold change)| > .5 and adjust p value < .05] ( Figure S1B). To our interest, several pathways related to ER function, such as response to ER stress and protein processing in ER, ranked top according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses ( Figure 1A and B). Enrichment of UPR and MYC targets was verified by Hallmark Gene Set Enrichement Analysis (GSEA) (Figures 1C and S1C), mirroring the recently established co-activation of UPR and MYC in multiple cancers. 4,8 Meanwhile, transcriptomic analyses on 24 matched tumour and normal tissues collected in our hospital (hereafter referred to as Zhengzhou cohort) enriched similar Hallmark pathways such as protein secretion, UPR and MYC signalling ( Figure 1D and E). Consistently, immunohistochemistry observed markedly elevated level of GRP78, and nuclear localisation of canonical UPR transcription factors XBP1s, ATF4, and ATF6 in tumour foci compared to normal tissues ( Figure 1F). By overlapping a repertoire of previously described UPR-related genes [4][5][6][7] (Table S2) with DEGs in the GSE99671, GSE126209 and Zhengzhou cohorts, we acquired 14 genes with significantly aberrant expression in OS (Figures S1D and S2A), defined as the OSspecific UPR gene signature.
Based on this signature, we constructed a set of scoring system 9 to quantify the UPR activity of each tumour (termed as UPR score) and conducted unsupervised consensus clustering 10 to classify different molecular features and prognosis. Interestingly, patients from GSE21257 were clustered into two subtypes with notable difference in global gene expression, such as genesets related to ER biology and UPR, as well as in overall and progression-free survival (Figures 2 and S2B-E). Likewise, this protocol led to optimal bifurcation of patients in both the TARGET OS and TCGA sarcoma datasets ( Figures 2C and D and S2F and G).
From a translational standpoint, we were further interested in developing a prognostic signature consisting of a handful of genes with higher power. Based on the classification strategy above, we applied Kaplan-Meier survival analyses coupled with univariate Cox regression to GSE21257 and TARGET cohorts and identified 21 candidate DEGs (Table S3). Using GSE21257 as discovery dataset, LASSO Cox regression based on overall survival and patient status established a linear model as follows: risk score = 2ˆ(0.4528 × expression level of NOP58 − 0.2303 × expression level of ALOX5AP + 0.2209 × expression level of MYC + 0.0828 × expression level of LGR4 + 0.0209 × expression level of GADD45GIP1) ( Figure  S3A). Subsequent survival analyses confirmed that the overall and progression-free survival of the high-risk subgroup was significantly shorter than the low-risk subgroup ( Figure 3A). Receiver Operator Characteristic (ROC) curve  analyses indicated that the 1-, 6-, and 12-year area under curve (AUC) values were .83, .83 and .82, respectively, for overall survival, while .83, .80 and .86 for progressionfree survival ( Figure S3B). Of these five genes, the expression of NOP58, MYC, LGR4, and GADD45GIP1 was significantly higher in the high-risk subgroup, whereas that of ALOX5AP was conversely profiled ( Figure 3B). Importantly, the level of NOP58 and ALOX5AP, and their correlation with OS pathology was validated in independent tissue microarrays ( Figure 3C-E; Table S4). Subsequent analyses of TARGET OS and TCGA sarcoma cohorts as validation datasets similarly subtyped patients with distinct status of risk scores, UPR activity, molecular charateristics and survival outcomes ( Figures S3C-G and S4A).
Additionally, we observed significant enrichment of multiple immune-relevant gene signatures according to pathway enrichment analyses ( Figures 4A and S4B-D). In fact, all the high-risk subgroups across different datasets uniformly showed lower stromal and immune scores, but higher tumour purity and stemness compared to the lowrisk subgroups ( Figures 4B and S5A-D). Dissection of immune infiltration by CIBERSORT uncovered significantly higher proportion of CD8 + T cells, monocytes and M2 macrophages, and lower proportion of memory resting CD4 + T cells and M0 macrophages in the low-risk subgroup ( Figures 4C and S5E and F). Interestingly, the level of several immune checkpoints was markedly higher in the low-risk, including PD-L2, CD86, TNFRSF14, CD4 and LAG3 ( Figures 4D and S6A-C). Submap analyses confirmed that the low-risk subtype was more likely to respond to anti-PD1 therapy ( Figures 4E and S6D and E), which warrants future investigation.
In conclusion, our study underlines that UPR activation is a common molecular feature of OS, and offers a novel prognostic gene signature refined from this perspective with translational value ( Figure 4F).

A C K N O W L E D G E M E N T S
This work was supported by National Natural Science Foundation of China (81802546 and 81972752); Fundamental Research Funds for Central Universities (2019kfyXJJS033); Young Talents in Health (HNSWJW-2020027) and Young Innovative Talents in Health Science (2020-167-1-4-5) of Henan Province.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.